The endemic forms of dengue virus that infect humans today resulted from the zoonotic transmission of four different dengue viruses that infect old world monkeys in Asia and Africa. These non-human primate dengue viruses (aka sylvatic dengue) continue to spill over into human populations, and will occasionally cause local dengue epidemics. Despite these obvious threats to public health, little is known about what adaptive barriers to viral replication exist during transmissions from monkey reservoir hosts to humans. It is well established that the innate immune system is the major barrier to cross-species transmission of dengue virus. Specifically, if a host is able to mount a robust interferon response upon infection, the virus will be unable to replicate. One of the nonstructural proteins from endemic, human-adapted Dengue, NS2B3, has been shown to antagonize the interferon response in human cells. Recently, it was shown that this antagonism could be explained by NS2B3s ability to cleave a human antiviral protein: the Stimulator of INterferon Genes (STING). I have shown that STING has been undergoing rapid evolution in primates, and this evolution has allowed for STING to avoid cleavage by NS2B3. This suggests that the evolution of STING may have been shaped by antagonism by viruses, and the barriers to cross species transmission are partially due to this antiviral protein. The goal of this proposal is to determine how the evolution of STING has contributed to barriers to sylvatic dengue spillover. The specific aims of this research proposal are to: 1) Determine how the evolution of STING has created species barriers for human endemic strains of dengue. 2) Determine the role of NS2B3 in extending or changing the host range of sylvatic dengue. 3) Determine if a human dengue strain can adapt to replicate in the presence of new world monkey STING. Uncovering the details that govern these transmission events could help in predicting which sylvatic strains have the potential to emerge as new endemic forms of the virus. Furthermore, knowing the factors responsible for species-tropism of the virus may help in developing an effective animal model for testing antiviral therapies, treatment protocols, and vaccines.